QC Report

	general
Report generated at	2025-02-25 03:26:49
Title	AdrenalGland
Description	ATAC-seq data from alternative Human ENCODE adrenal gland data processed in 2025
Pipeline version	v2.2.0
Pipeline type	atac
Genome	hg38
Aligner	bowtie2
Sequencing endedness	{'rep1': {'paired_end': True}, 'rep2': {'paired_end': True}}
Peak caller	macs2

Alignment quality metrics

SAMstat (raw unfiltered BAM)

	rep1	rep2
Total Reads	685308692	64080026
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	675281410	63376096
Mapped Reads (QC-failed)	0	0
% Mapped Reads	98.5	98.9
Paired Reads	685308692	64080026
Paired Reads (QC-failed)	0	0
Read1	342654346	32040013
Read1 (QC-failed)	0	0
Read2	342654346	32040013
Read2 (QC-failed)	0	0
Properly Paired Reads	664791842	62244114
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	97.0	97.1
With itself	671465730	63057354
With itself (QC-failed)	0	0
Singletons	3815680	318742
Singletons (QC-failed)	0	0
% Singleton	0.6	0.5
Diff. Chroms	202732	13939
Diff. Chroms (QC-failed)	0	0

Marking duplicates (filtered BAM)

	rep1	rep2
Unpaired Reads	0	0
Paired Reads	294098350	23557643
Unmapped Reads	0	0
Unpaired Duplicate Reads	0	0
Paired Duplicate Reads	107254561	10263652
Paired Optical Duplicate Reads	1073256	5312
% Duplicate Reads	36.4689	43.568200000000004

Filtered with samtools flag 1804 (samtools view -F 1804):

read unmapped (0x4)
mate unmapped (0x8, for paired-end)
not primary alignment (0x100)
read fails platform/vendor quality checks (0x200)
read is PCR or optical duplicate (0x400)

Fraction of mitochondrial reads (unfiltered BAM)

	rep1	rep2
Rn = Number of Non-mitochondrial Reads	552849420	32888947
Rm = Number of Mitochondrial Reads	133703666	33619613
Rm/(Rn+Rm) = Frac. of mitochondrial reads	0.19474628943696862	0.5054930222515718

SAMstat (filtered/deduped BAM)

	rep1	rep2
Total Reads	370410392	24993228
Total Reads (QC-failed)	0	0
Duplicate Reads	0	0
Duplicate Reads (QC-failed)	0	0
Mapped Reads	370410392	24993228
Mapped Reads (QC-failed)	0	0
% Mapped Reads	100.0	100.0
Paired Reads	370410392	24993228
Paired Reads (QC-failed)	0	0
Read1	185205196	12496614
Read1 (QC-failed)	0	0
Read2	185205196	12496614
Read2 (QC-failed)	0	0
Properly Paired Reads	370410392	24993228
Properly Paired Reads (QC-failed)	0	0
% Properly Paired Reads	100.0	100.0
With itself	370410392	24993228
With itself (QC-failed)	0	0
Singletons	0	0
Singletons (QC-failed)	0	0
% Singleton	0.0	0.0
Diff. Chroms	0	0
Diff. Chroms (QC-failed)	0	0

Filtered and duplicates are removed. Subsampling with atac.subsample_reads is not done in alignment steps. Nodup BAM is converted into a BED type (TAGALIGN) later and then TAGALIGN is subsampled with such parameter in the peak-calling step.

Fragment length statistics (filtered/deduped BAM)

	rep1	rep2
Fraction of reads in NFR	0.584658071356412	0.6681063190700142
Fraction of reads in NFR (QC pass)	True	True
Fraction of reads in NFR (QC reason)	OK	OK
NFR / mono-nuc reads	2.4207123440163403	3.2122539176994622
NFR / mono-nuc reads (QC pass)	False	True
NFR / mono-nuc reads (QC reason)	out of range [2.5, inf]	OK
Presence of NFR peak	True	True
Presence of Mono-Nuc peak	True	True
Presence of Di-Nuc peak	True	True

Open chromatin assays show distinct fragment length enrichments, as the cut sites are only in open chromatin and not in nucleosomes. As such, peaks representing different n-nucleosomal (ex mono-nucleosomal, di-nucleosomal) fragment lengths will arise. Good libraries will show these peaks in a fragment length distribution and will show specific peak ratios.

NFR: Nucleosome free region

Sequence quality metrics (filtered/deduped BAM)

Open chromatin assays are known to have significant GC bias. Please take this into consideration as necessary.

Annotated genomic region enrichment

	rep1	rep2
Fraction of Reads in universal DHS regions	0.6499729737604122	0.2775773501526093
Fraction of Reads in blacklist regions	0.0005192510905579561	0.002469909048963183
Fraction of Reads in promoter regions	0.3172393608222525	0.1002065439486248
Fraction of Reads in enhancer regions	0.3933251986083587	0.2870773235053911

Signal to noise can be assessed by considering whether reads are falling into known open regions (such as DHS regions) or not. A high fraction of reads should fall into the universal (across cell type) DHS set. A small fraction should fall into the blacklist regions. A high set (though not all) should fall into the promoter regions. A high set (though not all) should fall into the enhancer regions. The promoter regions should not take up all reads, as it is known that there is a bias for promoters in open chromatin assays.

Library complexity quality metrics

Library complexity (filtered non-mito BAM)

	rep1	rep2
Total Fragments	246285728	12897991
Distinct Fragments	191613673	12504946
Positions with Two Read	26182209	314948
NRF = Distinct/Total	0.778014	0.969527
PBC1 = OneRead/Distinct	0.814904	0.974088
PBC2 = OneRead/TwoRead	5.963853	38.675975

Mitochondrial reads are filtered out by default. The non-redundant fraction (NRF) is the fraction of non-redundant mapped reads in a dataset; it is the ratio between the number of positions in the genome that uniquely mapped reads map to and the total number of uniquely mappable reads. The NRF should be > 0.8. The PBC1 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with AT LEAST one read pair. PBC1 is the primary measure, and the PBC1 should be close to 1. Provisionally 0-0.5 is severe bottlenecking, 0.5-0.8 is moderate bottlenecking, 0.8-0.9 is mild bottlenecking, and 0.9-1.0 is no bottlenecking. The PBC2 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with EXACTLY two read pairs. The PBC2 should be significantly greater than 1.

Fragment: read for a single-ended dataset, pair of reads for a paired-ended dataset
NRF: non redundant fraction
PBC1: PCR Bottleneck coefficient 1
PBC2: PCR Bottleneck coefficient 2
PBC1 is the primary measure. Provisionally

0-0.5 is severe bottlenecking
0.5-0.8 is moderate bottlenecking
0.8-0.9 is mild bottlenecking
0.9-1.0 is no bottlenecking

Replication quality metrics

IDR (Irreproducible Discovery Rate) plots

Reproducibility QC and peak detection statistics

	overlap	idr
Nt	132818	44366
N1	277262	190293
N2	76934	30350
Np	276831	190049
N optimal	276831	190049
N conservative	132818	44366
Optimal Set	pooled-pr1_vs_pooled-pr2	pooled-pr1_vs_pooled-pr2
Conservative Set	rep1_vs_rep2	rep1_vs_rep2
Rescue Ratio	2.0842882741797046	4.283663165487085
Self Consistency Ratio	3.6038942470169237	6.26995057660626
Reproducibility Test	fail	fail

Reproducibility QC

N1: Replicate 1 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep1 reads)
N2: Replicate 2 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep2 reads)
Ni: Replicate i self-consistent peaks (comparing two pseudoreplicates generated by subsampling RepX reads)
Nt: True Replicate consistent peaks (comparing true replicates Rep1 vs Rep2)
Np: Pooled-pseudoreplicate consistent peaks (comparing two pseudoreplicates generated by subsampling pooled reads from Rep1 and Rep2)
Self-consistency Ratio: max(N1,N2) / min (N1,N2)
Rescue Ratio: max(Np,Nt) / min (Np,Nt)
Reproducibility Test: If Self-consistency Ratio >2 AND Rescue Ratio > 2, then 'Fail' else 'Pass'

Number of raw peaks

	rep1	rep2
Number of peaks	299736	218761

The number of peaks is capped at 300000
Peaks are called from macs2 with p-val threshold 0.01

Peak calling statistics

Peak region size

	rep1	rep2	idr_opt	overlap_opt
Min size	150.0	150.0	150.0	150.0
25 percentile	297.0	154.0	502.0	334.0
50 percentile (median)	543.0	215.0	776.0	590.0
75 percentile	932.0	353.0	1159.0	978.0
Max size	3603.0	2122.0	3598.0	3598.0
Mean	677.8524968639069	320.4930814907593	872.405390188846	717.5627621184044

Enrichment / Signal-to-noise ratio

TSS enrichment (filtered/deduped BAM)

	rep1	rep2
TSS enrichment	26.084253634636482	14.011771178374902

Open chromatin assays should show enrichment in open chromatin sites, such as TSS's. An average TSS enrichment in human (hg19) is above 6. A strong TSS enrichment is above 10. For other references please see https://www.encodeproject.org/atac-seq/

Jensen-Shannon distance (filtered/deduped BAM)

	rep1	rep2
AUC	0.09818536463904944	0.22365774548383893
Synthetic AUC	0.49798566493745205	0.49205019167743685
X-intercept	0.10402589302135895	0.21046876124523203
Synthetic X-intercept	0.0	4.6889282558342433e-135
Elbow Point	0.8711226439989445	0.6257666749298297
Synthetic Elbow Point	0.5029721236463212	0.5136372569637959
Synthetic JS Distance	0.6374358077643321	0.3230172010275505

Peak enrichment

Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

	rep1	rep2	rep1-pr1	rep2-pr1	rep1-pr2	rep2-pr2	pooled	pooled-pr1	pooled-pr2
Fraction of Reads in Peaks	0.6018843445407439	0.17848302748248446	0.5954732771104327	0.22724323564767224	0.5950068161154615	0.2282485479666732	0.575331186902133	0.5690982444723192	0.5686598772160963

FRiP for overlap peaks

	rep1_vs_rep2	rep1-pr1_vs_rep1-pr2	rep2-pr1_vs_rep2-pr2	pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks	0.44673011845465654	0.5940709298458343	0.1150220771802666	0.567628799149588

FRiP for IDR peaks

	rep1_vs_rep2	rep1-pr1_vs_rep1-pr2	rep2-pr1_vs_rep2-pr2	pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks	0.28312243069499465	0.5497413798260822	0.07460468891813414	0.5246375766615389

For macs2 raw peaks:

repX: Peak from true replicate X
repX-prY: Peak from Yth pseudoreplicates from replicate X
pooled: Peak from pooled true replicates (pool of rep1, rep2, ...)
pooled-pr1: Peak from 1st pooled pseudo replicate (pool of rep1-pr1, rep2-pr1, ...)
pooled-pr2: Peak from 2nd pooled pseudo replicate (pool of rep1-pr2, rep2-pr2, ...)

For overlap/IDR peaks:

repX_vs_repY: Comparing two peaks from true replicates X and Y
repX-pr1_vs_repX-pr2: Comparing two peaks from both pseudoreplicates from replicate X
pooled-pr1_vs_pooled-pr2: Comparing two peaks from 1st and 2nd pooled pseudo replicates